3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctanesulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

polyfluorosulphonic acid

IUPAC Name:

polyfluorosulphonic acid

Test material form:

liquid

Details on test material:

- Purity: 29.8% solid content

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500, and 5000 μg/plate. Since the test item was freely soluble and almost non-toxic in the preliminary test, the highest dose-level for the main test was 5000 μg/plate, according to the criteria specified in the international guidelines.

Vehicle / solvent:

Distilled water

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-Anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: The test substance was tested in a preliminary test and 2 mutagenicity experiments. The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

Direct plate incorporation method: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL), and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

Preincubation method: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3 plates/dose

DETERMINATION OF CYTOTOXICITY: The evaluation of the toxicity in the preliminary toxicity test was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

- OTHER: Rertants were scored with an automatic counter. The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found satisfactory.

Evaluation criteria:

This study is considered valid if the following criteria are fully met:
. the number of revertants in the vehicle controls is consistent with our historical data
. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationhip was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number, of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle) were recorded.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test: No precipitate was observed in the Petri plates when scoring the revertants at all dose levels. Except for a moderate to strong thinning of the bacterial lawn noted in the TA102 strain without S9 mix at dose levels of ≥2500 µg/plate, no noteworthy toxicity was induced in the 3 strains used, with and without S9 mix.

Mutagenicity Experiments: No toxicity was noted towards all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Any other information on results incl. tables

Table 1 – TA 98 without S9 - Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		17	2	-----	16, 20, 16
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	13 21 15 13 15	0 6 4 3 5	0.7 1.2 0.9 0.7 0.9	13, 13, 13 28, 17, 18 11, 16, 19 16, 12, 10 10, 16, 19
2NF	0.5µg	159	27	9.2	177, 128, 171

 

Table 2 – TA 98 with S9 - Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		19	5	-----	22, 22, 14
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	17 24 30 26 27	4 6 5 5 1	0.9 1.3 1.6 1.4 1.4	22, 15, 14 26, 29, 18 25, 34, 31 31, 22, 26 28, 27, 26
2AM	2µg	1090	57	56.4	1103, 1139, 1028

 

Table 3 – TA 98 without S9 - Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		13	3	-----	11, 16, 13
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	17 14 13 19 21	4 5 1 2 7	1.2 1.1 1.0 1.4 1.5	20, 13, 17 20, 13, 10 14, 13, 12 20, 17, 20 26, 23, 13
2NF	0.5 µg	154	9	11.5	159, 143, 159

                       

Table 4 – TA 98 with S9 - Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		21	4	-----	26, 18, 19
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	20 27 25 25 20	4 8 7 3 4	1.0 1.3 1.2 1.2 0.9	23, 22, 16 30, 32, 18 25, 18, 31 24, 28, 22 24, 18, 17
2AM	2 µg	1002	44	47.7	1035, 952, 1020

Table 5 – TA 1535 without S9 – Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		10	4	-----	11, 6, 14
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	12 12 13 16 9	3 2 7 4 2	1.2 1.2 1.3 1.5 0.9	11, 16, 10 12, 14, 11 6, 14, 19 16, 20, 12 12, 8, 8
Sodium azide	1 µg	450	82	43.6	464, 362, 525

 

Table 6 – TA 1535 with S9 - Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		14	6	-----	7, 17, 18
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	15 12 12 12 12	3 1 2 1 5	1.0 0.9 0.9 0.9 0.9	14, 12, 18 13, 12, 12 10, 13, 13 12, 13, 11 16, 13, 7
2AM	2 µg	210	18	15.0	225, 216, 190

 

Table 7 - TA 1535 without S9 – Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		21	1	-----	22, 20, 20
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	19 19 32 19 20	5 4 6 1 3	0.9 0.9 1.5 0.9 1.0	13, 23, 20 23, 16, 17 35, 25, 35 20, 19, 18 23, 17, 19
Sodium azide	1 µg	428	29	20.7	436, 396, 452

 

Table 8 - TA 1535 with S9 – Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		13	4	-----	10, 11, 17
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	13 10 8 12 11	3 4 1 5 2	1.1 0.8 0.6 0.9 0.9	16, 13, 11 6, 13, 11 8, 7, 8 16, 6, 14 9, 12, 12
2AM	2 µg	109	33	8.6	74, 138, 116

Table 9 – TA 1537 without S9 - Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		6	1	-----	6, 6, 7
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	9 5 7 6 4	6 1 4 1 2	1.4 0.8 1.1 1.0 0.6	12, 2, 13 6, 5, 5 6, 11, 4 6, 7, 6 4, 5, 2
9AA	250 µg	455	96	71.9	553, 452, 361

 

Table 10 – TA 1537 with S9 - Replica 1

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		5	1	-----	6, 4, 4
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	8 8 7 11 10	3 3 1 5 3	1.8 1.6 1.6 2.4 2.1
2AM	2 µg	81	8	17.4	77, 90, 77

Table 11 – TA 1537 without S9 - Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		7	1	-----	7, 8, 7
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	8 7 6 5 7	3 8 1 1 3	1.1 1.0 0.9 0.7 1.0	11, 8, 6 4, 2, 16 7, 5, 7 5, 6, 4 11, 6, 5
9AA	50 µg	669	119	91.2	595, 606, 806

 

Table 12 – TA 1537 with S9 - Replica 2

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		5	4	-----	4, 2, 10
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	9 8 7 6 5	4 1 3 1 3	1.6 1.4 1.2 1.1 0.9	5, 13, 8 8, 7, 8 10, 4, 6 7, 5, 6 8, 2, 5
2AM	2 µg	80	11	15.0	93, 73, 74

Table 13 – TA 100 without S9

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		104	6	-----	102, 111, 99
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	106 111 93 114 112	9 4 9 12 4	1.0 1.1 0.9 1.1 1.1	105, 97, 115 114, 111, 107 86, 91, 103 125, 115, 102 109, 111, 117
Sodium azide	1µg	700	11	6.7	687, 703, 709

 

Table 14 – TA 100 with S9

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		126	18	-----	143, 127, 108
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	125 126 132 132 118	16 5 9 13 10	1.0 1.0 1.0 1.0 0.9	128, 140, 108 131, 121, 126 139, 122, 134 131, 145, 120 108, 120, 127
2AM	1µg	1599	252	12.7	1854, 1591, 1351

 

Table 15 – TA 102 without S9

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		249	35	-----	255, 211, 281
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	167 327 331 364 392	7 61 20 34 23	0.7 1.3 1.3 1.5 1.6	163, 175, 163 378, 345, 259 309, 340, 345 339, 350, 402 389, 370, 416
MMC	0.5µg	2234	64	9.0	2212, 2306, 2183

 

Table 16 – TA 102 with S9

 

Compound	Dose level per plate	Mean revertant colony counts	Standard deviation (SD)	Ratio treated/solvent	Individual revertant colony counts
Distilled Water		561	38	-----	568, 595, 520
Test substance (TS)	312.5µg 625µg 1250µg 2500µg 5000µg	567 525 557 545 572	16 43 37 0 26	1.0 0.9 1.0 1.0 1.0	582, 569, 550 575, 502, 498 522, 595, 555 545, 545, 545 600, 549, 567
2AM	10µg	4313	100	7.7	4413, 4213, 4312

2AM = 2-Anthramine

2NF = 2 -Nitrofluorene

9AA = 9-Aminoacridine

MMC = Mitomycin C

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic when tested in Salmonella typhimurium strains TA98, TA100, TA102, TA1535, and TA1537 in the presence or in the absence of a metabolic activation system (Aroclor-induced rat liver S9).

Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce reverse mutation in Salmonella typhimurium stains TA98, TA100, TA102, TA1535, and TA1537 in the presence or in the absence of a metabolic activation system [Aroclor-induced rat liver S9] (OECD Guideline No. 471 and EU Commission Directive No. 2000/32/EC, B13/14). A preliminary toxicity test was performed to define the dose levels of test substance to be used for the mutagenicity study. The test item was then tested in 2 independent experiment, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254). Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98, TA100, and TA102 were used. Each strain was exposed to 5 dose levels of the test item (3 plates/dose level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The selected treatment levels were 312.5, 625, 1250, 2500, and 5000 µg/plate for both mutagenicity experiments with and without S9 mix.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and almost non-toxic in the preliminary test, the highest dose level for the main test was 5000 µg/plate, according to the criteria specified in the international guidelines. No toxicity was noted toward all the strains used, both with and without S9 mix. The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the 5 strains. Under the experimental conditions, the test item does not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, with and without S9 mix up to 5000 µg/plate expressed as active material.